Blastomyces dermatitidis is a fungus found primarily in soil and is endemic throughout the Southeast, Southcentral and Upper Midwestern U.S. and in parts of Canada, India and Africa. Most cases of blastomycosis in the U.S. occur in the Ohio and Mississippi river valleys, the southeastern states, and around the Great Lakes (Bradsher, Chapman, et al., 2003). Blastomycosis typically starts in the lungs after inhalation of conidia or hyphal fragments causing pneumonia-like symptoms and may lead to disseminated disease if not diagnosed and treated early (Brandhorst, et al., 2005). Dogs are particularly vulnerable to infection, especially hunting dogs.
Upon entry into the host, B. dermatitidis undergoes a temperature-dependent phase transition into a pathogenic yeast form. Upon transition, yeast phase cells secrete and display on their surface BAD-1 (Blastomyces adhesin 1), a 120-kDa multi-functional protein that promotes adherence to macrophages by binding CD11b/CD18 (CR3) and CD14 (Newman, Chaturvedi, et al., 1995) and deviates host pro-inflammatory responses by suppressing tumor necrosis factor-α (TNF-α) (Finkel-Jimenez, Wüthrich, et al., 2001; Brandhorst, Finkel-Jimenez, et al., 2004) and inducing transforming growth factor-β (Finkel-Jiminez, Wüthrich, et al., 2002). Soluble BAD-1 released by wild-type yeast enters macrophages via CR3 receptor-mediated endocytosis, and this event has likewise been demonstrated to suppress tumor necrosis factor-α responses and control of the infection (Finkel-Jiminez, Wüthrich, et al., 2002).
A variety of techniques have been used to aid in the clinical diagnosis of blastomycosis. These include microscopic detection of characteristic broad-based budding yeast forms in body fluids or tissue biopsies, isolation and identification of the organism in culture, detection of B. dermatitidis-specific antigens in urine, and detection of specific immunologic responses to infection (Rippon, 1988; Mongkolrattanothai, Peev, et al., 2006). However, clinical tests for blastomycosis suffer from potential false positives due to cross-reactivity with common fungal antigens. One approach to reduce the number of false positives in blastomycosis detection is to use BAD-1, a protein unique to B. dermatitidis, as a biomarker for diagnosing blastomycosis infection.
To date, methods for isolating BAD-1 have relied on recombinant BAD-1 proteins, either full length or truncated, that display a 6×His tag and are purified on nickel affinity agarose columns (Finkel-Jimenez, Wüthrich, et al., 2001; Hogan, et al., 1995).